Designing a Hybrid Chain Reaction Probe for Multiplex Transcripts Assay with High-Level Imaging.

The hybrid chain reaction (HCR), an isothermal and enzyme-free amplification strategy, has found extensive use in fluorescent in situ hybridization (FISH) assays. However, the existing HCRs are limited, being time-consuming processes and low-efficiency imaging due to weak signal, significantly restricting their application in transcriptomic assays. To address the limitations, we developed nine orthogonal HCR hairpin-pair (hp) probes in this study to enable efficient signal amplification for multiplex assays. To enhance the efficiency and imaging quality of multiplex assays using these HCR probes, we employed two strategies. First, we coupled fluorescent molecules to HCR hairpins via disulfide bonds, facilitating easy removal through chemical cleavage. As a result, the workflow was greatly simplified. Second, we combined HCR with in situ rolling circle amplification (ISRCA), creating ISRCA-HCR, which achieved a 17-fold signal amplification. ISRCA-HCR demonstrated a high-level imaging capability for spatial cell type assays. This study shows the application for cell typing based on the developed HCR probes, enabling accurate and high-level signal amplification for multiplex FISH imaging. This provides an effective research tool for transcriptome and spatial cell type analysis.

Three-dimensional mapping of hepatic lymphatic vessels and transcriptome profiling of lymphatic endothelial cells in healthy and diseased livers.

Rationale: Hepatic lymphatics are essential for liver homeostasis and immune function. However, the 3D structure and spatial distribution of hepatic lymphatic vessels (LVs) need to be confirmed. Moreover, the molecular information of hepatic lymphatic endothelial cells (LyECs) needs to be further studied. The bottleneck is the lack of specific markers or labeling methods for hepatic lymphatic endothelial cells (LyECs) Methods: Here, we proposed a method for the spatiotemporal sequential injection of antibodies (STSI-Ab) to selectively label hepatic LyECs in vivo. In addition, we also developed an efficient hepatic LyEC sorting method and performed deep transcriptome sequencing on hepatic LyECs. Results: The STSI-Ab method achieved selective labeling of the mouse hepatic lymphatic network. Three-dimensional fluorescence imaging results of the STSI-Ab mouse liver lobe clearly showed that hepatic LVs entangled with the portal vein but were not present in the central vein. The imaging data inspired a novel hepatic lobule structure model with an added set of LVs in the portal area. Furthermore, deep transcriptome sequencing of isolated hepatic LyECs and Masson's trichrome staining results suggested that hepatic LyECs might be an important source of collagen fibers deposited in the portal area during the process of liver fibrosis and bile duct ligation (BDL). Conclusions: We proposed an STSI-Ab method for selectively labeling hepatic LVs, distinguishing the hepatic LVs from other vessels, and mapping their 3D structure. This study opens an avenue for understanding hepatic lymphatic structure and it will be very beneficial to the study of hepatic LyEC functions.

Cryo-fluorescence micro-optical sectioning tomography for volumetric imaging of various whole organs with subcellular resolution.

Optical visualization of complex microstructures in the entire organ is essential for biomedical research. However, the existing methods fail to accurately acquire the detailed microstructures of whole organs with good morphological and biochemical preservation. This study proposes a cryo-fluorescence micro-optical sectioning tomography (cryo-fMOST) to image whole organs in three dimensions (3D) with submicron resolution. The system comprises a line-illumination microscope module, cryo-microtome, three-stage refrigeration module, and heat insulation device. To demonstrate the imaging capacity and wide applicability of the system, we imaged and reconstructed various organs of mice in 3D, including the healthy tongue, kidney, and brain, as well as the infarcted heart. More importantly, imaged brain slices were performed sugar phosphates determination and fluorescence in situ hybridization imaging to verify the compatibility of multi-omics measurements. The results demonstrated that cryo-fMOST is capable of acquiring high-resolution morphological details of various whole organs and may be potentially useful for spatial multi-omics.

Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues. Despite the recent advances in FISH detection methods, it remains challenging to achieve high-level FISH imaging with a simple workflow. Here, we introduce a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA). Prepared lssDNAcs are used to develop AmpFISH workflows. In addition, we present its applications in different scenarios. AmpFISH shows the following advantages: 1) enhanced FISH signal-to-noise ratio (SNR) up to 160-fold compared with single-molecule FISH; 2) simultaneous detection of FISH signals and fluorescent proteins or immunofluorescence (IF) in tissues; 3) simple workflows; and 4) cost-efficiency. In brief, AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows.

Detection of Essential Tremor at the [Formula: see text]-Band.

Essential tremor (ET) is a neurological disorder characterized by rhythmic, involuntary shaking of a part or parts of the body. The most common tremor is seen in the hands/arms and fingers. This paper presents an evaluation of ETs monitoring based on finger-to-nose test measurement as captured by small wireless devices working in shortwave or [Formula: see text]-band frequency range. The acquired signals in terms of amplitude and phase information are used to detect a tremor in the hands. Linearly transforming raw phase data acquired in the [Formula: see text]-band were carried out for calibrating the phase information and to improve accuracy. The data samples are used for classification using support vector machine algorithm. This model is used to differentiate the tremor and nontremor data efficiently based on secondary features that characterize ET. The accuracy of our measurements maintains linearity, and more than 90% accuracy rate is achieved between the feature set and data samples.